侵略性牙周炎病患之多形核嗜中性白血球功能異常及IL-8基因多形性變化

Abstract

牙周炎的產生是一種複雜且多因子的過程，牙周的破壞程度主要是受到細菌牙菌斑形成與宿主免疫系統所影響。在先天性免疫中，吞噬細胞為人體防禦系統的第一道防線，其中多形核嗜中性白血球 (polymorphonuclear neutrophils, PMNs)之功能異常，往往會影響牙周炎的嚴重性。在過去，早發性牙周炎 (early-onset periodontits)是嚴重牙周炎的其中一種，早發性牙周炎病患者和其家人有相當高比率具有周邊血液之PMNs的不正常趨化反應 (chemotaxis)和其他缺陷，如: 氧自由基過分的釋出，因而導致牙周組織的快速破壞。另一方面，細胞白介素-8 ( interleukin-8, IL-8)對多形核嗜中性白血球是一種強有力的趨化引誘劑。而且，在其它種類的發炎疾病中，IL-8的啟動子片段基因表現已被証實為和疾病的易感受性基因有關，可能扮演一個致病的角色。在英國，有報告指出不同的IL-8-25(A→T)基因型，會影響IL-8血清中的濃度。在非常嚴重的牙周炎病人，其PMNs不正常趨化反應的特性，是由於PMNs本身趨化能力缺失或是因環境因素 (如:吸菸，等)所影響，時至今日仍有所爭論。本研究希望探討侵略型牙周炎 (AgP)、慢性牙周炎 (CP)和牙周健康者 (H)的PMNs趨化異常反應機制與氧自由基釋出在牙周破壞上可能的角色。並進一步研究華人在不同牙周炎與IL-8啟動子片段基因多型性，及其與IL-8血中濃度之間可能的相關性。 受測病患是由國立台灣大學附設醫院牙周病科臨床門診的病人群中篩檢獲得，並抽取其靜脈血液檢查。共有39位AgP、19位CP和34位H參與PMNs的功能檢測。依Boyum所提出的方法，藉由Ficoll-Hypaque梯度離心分離出PMNs，再用Polycarbonate Membrane Transwell&reg; Inserts評估PMNs的趨化反應。所使用的趨化引誘劑 (chemoattractants)分別為IL-8和N-fMLP，並用RPMI 1640作為平行的對照組。氧自由基的釋出是用Phorbol myristate acetate刺激全血後，以luminolenhanced chemiluminescence的方式測量。IL-8啟動子片段基因多型性是藉由DNA萃取、聚合酶鏈反應，和兼性Mfe I限制內切酶等步驟，從38位AgP、20位CP，和36 位H鑑定出來。IL-8啟動子片段基因多型性是一個IL-8-251A或被取代成IL-8-251T。這些受測者的周邊血液樣本以等體積RPMI 1640稀釋後，而且用或不用脂多醣 (LPS)刺激。之後，用Chemiluminescent Immunoassay測量受測者血中所生成的IL-8濃度，並用全白血球計數校正。PMNs的功能檢測和IL-8血中濃度實驗結果是皆用ANOVA (變數分析)和複迴歸去做統計分析。此外，牙周炎和IL-8啟動子片段基因多型性之間的關聯性是以Chi-Square test或Fisher’s exact test去檢測。 ANOVA結果和複迴歸統計結果相似。根據複迴歸統計分析結果，PMNs的趨化反應在IL-8誘導下，CP有較低落的情形 (b = -0.31, p < 0.01); 但在N-fMLP誘導下，AgP呈現較活躍 (b = 15.27, p = 0.057)。然而，在氧自由基的釋出量中，AgP, CP, 和H三組之間無顯著差異。此外，無LPS刺激時，具IL-8-251基因型AT (b = -0.54, p = 0.04)或IL-8-251對偶基因型A (b = -0.54, p = 0.034)的人，血中IL-8濃度會較低。而用LPS刺激時，AgP和CP的IL-8血中濃度會較高 (b = 0.22, 0.62; p = 0.03, < .0001; CP > AgP > H)。牙周炎和IL-8-251基因多型性之間無相關性。 推測在由細菌萃取出的N-fMLP誘導下，AgP的PMNs遷移數目較多，因此引發其牙周組織的快速破壞; 當使用人類IL-8作為趨化引誘劑時，CP的PMNs遷移數目較少，因此CP的牙周破壞較緩慢。由PMA刺激所釋出的氧自由基，不是引起牙周快速破壞的主要原因。沒有LPS刺激時，IL-8-251基因型AT或IL-8-251對偶基因型A，對血中IL-8濃度皆有負影響，但沒有證據顯示，IL-8-251基因多型性會影響牙周的破壞速率。當LPS刺激時，AgP和CP血中的IL-8濃度會較牙周正常者高。已知IL-8影響PMNs的遷移相當大，所以當IL-8濃度較高時，可能會使PMNs的趨化反應過分活化，而與AgP的快速牙周破壞有關。但由於CP的PMNs對IL-8的趨化反應低落，所以即使IL-8濃度提高，也可能不會促使其牙周快速破壞。

Periodontitis is a complex, multi-factorial process affected by bacterial plaque-components and host defense mechanisms. Phagocytic cells in innate immunity provide the host with the first line of defense against acute bacterial and fungal infections, and the functional disorders of polymorphonuclear neutrophils (PMNs) often determine the seriousness of periodontal diseases. In the past, early-onset periodontits (EOP) was a severe peridontitis. A high percentage of the patients and their families with a diagnosis of EOP were reported to exhibit an abnormal neutriphil chemotaxis and other defects, such as overproduction of oxygen radicals, and lead to rapidly periodontal destruction. On the other hand, IL-8 acts as a potent chemoattractant for PMNs. IL-8 promoter gene expression is demonstrated to be a disease susceptibility gene in a variety of inflammatory diseases and may play a pathogenic role in disease. Different IL-8-251(A→T) gene genotypes affect IL-8 concentration in serum are reported in UK. Until now, whether the trait of abnormal chemotactic response of PMNs in severe periodontitis patient is due to an intrinsic defect of PMN or is environmentally influenced (e.g. smoking) still remains controversial. The aim of the present study is to compare the chemotaxis function of PMNs and the amount of oxygen radicals released from peripheral blood among aggressive periodontitis patients (AgP), chronic periodontitis patients (CP) and control subjects with healthy periodontium (H). Besides, this study will further investigate into the possible correlation among types of periodontitis, IL-8 promoter gene polymorphism and IL-8 concentration in blood in Taiwan. In this experiment, the patients were selected from Periodontal Department of National Taiwan University Hospital and their venous blood was taken for examination. There were 39 subjects of AgP, 19 subjects of CP, and 34 control subjects participating in the PMNs function studies. PMNs were separated from venous blood by Ficoll-Hypaque gradient centrifugation which was proposed by Boyum, and the chemotaxis of PMNs was assessed in Polycarbonate Membrane Transwell&reg; Inserts. IL-8 and N-fMLP (N-formylmethionine-leucyl-phenylanine) were used as chemoattractants, and RPMI 1640 as a parallel control in the assessment of PMN chemotaxis. The generation of free oxygen radicals was measured with luminal-enhanced chemiluminescence (CL) after whole blood was activation by Phorbol myristate acetate (PMA). Genetic polymorphisms of the IL-8 promoter region were identified by DNA extraction, polymerase chain reaction, and facultative Mfe I restriction endonuclease from 38 subjects of AgP, 20 subjects of CP, and 36 control subjects. IL-8 promoter gene polymorphism is a IL-8-251A or IL-8-251T by substituted. All the subjects’ whole blood was diluted with an equal volume of RPMI 1640, and stimulated with or without lipopolysaccharide (LPS). The IL-8 concentration in serum of the subjects was measured by Chemiluminescent Immunoassay and corrected with the total white cell count. Two statistical analyses ANOVA (analysis of variance) and multiple linear regression analysis of PMN functions study and IL-8 concentration in serum studies were used in our study. In addition, Chi-Square test or Fisher’s exact test assayed the association between periodontitis and IL-8 promoter gene polymorphism. The results of ANOVA and multiple linear regression analysis are similar. According to the result of multiple regression analysis, PMNs from CP shows depressed chemotactic response to IL-8 (b = -0.31, p < 0.01), but PMNs from AgP shows active chemotactic response to N-fMLP (b = 15.27, p = 0.057). However, no significant differences in release of oxygen radicals among the three groups (AgP, CP, H) are found. Besides, the concentration of IL-8 in the serum without LPS stimulation is lower in both IL-8 -251AT genotype subjects (b = -0.54, p = 0.04) and IL-8 -251A allele type subjects (b = -0.54, p = 0.034). The concentration of IL-8 in the serum with LPS stimulation is higher in AgP and CP subjects (b = 0.22, 0.62; p = 0.03, < .0001; CP > AgP > C). There is no correlation between periodontitis and IL-8 promoter gene polymorphism. We speculate that the number of PMNs migrating under N-fMLP as chemoattractant from bacteria is larger in AgP, and spark off the faster periodontal destruction in AgP. In CP, the number of PMNs migrating under human IL-8 as chemoattractant from host is smaller which thus results in slower periodontal destruction in CP. The release of oxygen radicals stimulated by PMA isn’t the major cause of rapid periodontal destruction. Whether IL-8 -251AT genotype or IL-8 -251A allele type has a negative effect on the production of IL-8 in serum without LPS stimulation, but IL-8 promoter gene polymorphism can’t affect the rate of periodontal destruction. AgP and CP subjects have higher concentration of IL-8 in serum with LPS stimulation. Furthermore, since IL-8 has large influences on PMN migration, we can infer that higher concentration of IL-8 may lead to hyperactive PMN chemotaxis, and be related to rapid periodontal destruction in AgP. On the other hand, CP has depressed chemotactic response to IL-8, and therefore the high concentration IL-8 may not lead to rapid periodontal destruction.